【作者】 刘浩；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2016， 博士

【摘要】 骨质疏松是严重危害人类健康的骨代谢疾病,而且发病率随着人口老龄化逐渐升高,目前治疗骨质疏松的药物在有效性和安全性方面存在一定的问题,迫切需要研发安全而有效的防治手段,若能针对尚未发病和症状较轻的人群开发具有一定疗效且安全无副作用的功能食品,将是一条很好防治骨质疏松的途径。玛咖(Lepidium meyennii Walp.)具有改善更年期综合症等多种生理功效,前期研究发现其具有较好的抗骨质疏松效果,但其抗骨质疏松的功效物质基础和作用机制尚不清楚,严重阻碍了玛咖抗骨质疏松功能产品的开发和利用。本文在前期研究基础上,通过构建玛咖次生代谢物数据库,并以诱导骨质疏松发病的关键因子雌激素受体为靶点,采用虚拟筛选技术寻找玛咖化合物库中潜在的抗骨质疏松活性成分,并通过反向对接技术预测活性成分可能的作用途径,进一步在细胞和动物水平上进行活性评价,取得如下创新性结果：(1)通过分子对接预测,发现玛咖酰胺最有可能是玛咖抗骨质疏松活性物质,其中,N-benzyl-palmitamide是结合能力最好的结构之一。通过文献调查构建了玛咖次生代谢物数据库,并利用AutoDock Vina将其中的化合物与雌激素受体(PDB号：1 GWR)进行分子对接,其结合自由能分布在-7.7到-2.1 kcal/mol之间,其中,玛咖酰胺类化合物的结合能最低,在-7.7到-7.0 kcal/mol之间,表明玛咖酰胺是最有可能的抗骨质疏松活性物质；进一步研究玛咖酰胺结构与雌激素受体结合能力之间的规律,发现苯基、酰胺键和碳链长度大于15的脂肪长链是维持玛咖酰胺类物质结合雌激素受体能力的关键结构,其中,N-benzyl-palmitamide是结合能力最好的结构之一(2)通过反向对接预测,发现N-benzyl-palmitamide可通过与细胞视黄酸结合蛋白、视黄醇结合蛋白、视黄酸受体、甲状腺素运载蛋白等视黄酸信号通路相关蛋白,以及维生素D3受体和碳酸酐酶Ⅱ相互作用,调节骨代谢。通过采用PharmMapper进行反向预测,发现N-benzyl-palmitamide在人体内的潜在作用靶点为细胞视黄酸结合蛋白2、甲状腺素运载蛋白、含黄素胺氧化酶B、视黄醇结合蛋白4、视黄酸受体β、维生素D3受体、α-生育酚转移蛋白、血清蛋白和碳酸酐酶Ⅱ,表明N-benzyl-palmitamide可能对视觉系统、神经系统、免疫系统、生殖系统、骨骼系统和基础代谢起到调节作用。其中,视黄醇结合蛋白、细胞视黄酸结合蛋白、视黄酸受体、甲状腺素运载蛋白、维生素D3受体和碳酸酐酶Ⅱ均与骨代谢密切相关,进一步表明玛咖酰胺可能具有抗骨质疏松活性。(3)通过细胞模型评价,证实N-benzyl-palmitamide具有促成骨作用。通过采用MC3T3-E1 Subclone14细胞模型评价N-benzyl-palmitamide对成骨细胞的影响,发现N-benzyl-palmitamide可以促进MC3T3-E1细胞增殖、分化和基质矿化,具有促成骨作用；进一步探索其作用机理,发现N-benzyl-palmitamide可通过提高雌激素受体以及成骨关键因子骨形态发生蛋白2(BMP-2)、核心结合因子1(Cbfal)、Ⅰ型胶原(COL1)和碱性磷酸酶(ALP)的大量表达,促进成骨作用。(4)通过动物模型评价,进一步证实N-benzyl-palmitamide具有抗骨质疏松作用。通过构建视黄酸致骨质疏松大鼠模型,研究了N-benzyl-palmitamide在动物水平的抗骨质疏松作用效果,发现N-benzyl-palmitamide可以提高视黄酸致骨质疏松大鼠的股骨和脊椎骨密度,增加其骨灰分和骨钙含量,改善骨组织结构形态,具有较好的抗骨质疏松效果；机理探索发现,N-benzyl-palmitamide可以通过促进ALP活性,拮抗视黄酸引起的雌激素水平下降和抗酒石酸性磷酸酶(TRAP)活性上升,实现其抗骨质疏松作用的。(5)比较研究了含有N-benzyl-palmitamide的玛咖醇提物与N-benzyl-palmitamide单独作用效果,发现在含有相同剂量N-benzyl-palmitamide条件下,玛咖醇提物在促进成骨细胞增殖、提高股骨和脊椎骨密度、以及改善骨组织物理参数和形态结构等方面的效果,与N-benzyl-palmitamide单独作用相似,在促进ALP活性、提高骨灰分和骨钙含量方面的效果显著优于N-benzyl-palmitamide单独作用,表明玛咖中N-benzyl-palmitamide是重要的促成骨作用活性成分,同时也含有其他具有一定抗骨质疏松作用或协同作用的活性成分。在实验使用剂量下,玛咖醇提物和N-benzyl-palmitamide均没有表现出子宫增重等明显的不良反应。综上,本文采用虚拟筛选技术结合MC3T3-E1成骨细胞以及视黄酸致骨质疏松大鼠模型的活性评价,发现玛咖中的N-benzyl-palmitamide具有抗骨质疏松活性；其通过提高雌激素受体和成骨关键因子骨形态发生蛋白2、核心结合因子1、Ⅰ型胶原和碱性磷酸酶的表达,促进骨形成过程；拮抗视黄酸引起的雌激素水平降低和抗酒石酸性磷酸酶活性上升,抑制骨吸收过程,最终起到抗骨质疏松作用。更多还原

【Abstract】 Osteoporosis is a bone metabolic disease, which causing disability to the elderly and enormous long-term care expenses. Along with the increased population of elderly persons, the demand for preventing osteoporosis products has grown rapidly. However, there are many problems in efficacy and safety of anti-osteoporosis drug. Thus, developing an effective and safe functional food will be an effective way for the people who had not yet been onset and mild symptoms.Maca(Lepidium meyennii Walp.) has many physiological functions such as improving menopause syndrome and preventing postmenopausal osteoporosis. But the efficacy material base of maca on preventing postmenopausal is unclear, severely hindering the exploitation and application of maca anti-osteoporosis products. In this paper, maca compound library was built, and potential active ingredients to estrogen receptors in the library were discovered by virtual screening, and then its action target were predict through reverse docking, further were evaluation on cell and animal models. The innovative results as follow:(1) Macamides were screened out as active constituent of maca in preventing osteoporosis by using virtual screening against estrogen receptor, N-benzyl-palmitamide was one of the best structures. Maca compound library was constructed by literature survey. By AutoDock Vina, the compounds were screened out for estrogen receptor (PDB:1 GWR), which free binding energy were between 7.7 and 2.1 kcal/mol. The binding energy of macamides were lowest, between 7.7 and 7.0 kcal/mol, which suggest macamides were most likely as maca anti-osteoporosis active substances. Furthermore, phenyl, amide bond and the carbon chain length is greater than 15 were key structures for macamides to bind with estrogen receptor. Among macamides, N-benzyl-palmitamide was one of the best structures.(2) Through reverse docking, there was found that N-benzyl-palmitamide could regulate bone metabolism by affectting retinoic acid signaling pathways related protein, such as cellular retinoic acid-binding protein, retinol binding protein, retinoic acid receptors and transthyretin, and vitamin D3 receptor and carbonic anhydrase 2. The targets in the body of N-benzyl-palmitamide were found by PharmMapper, including cellular retinoic acid-binding protein 2, transthyretin, amine oxidase [flavin-containing] B, retinol-binding protein 4, retinoic acid receptor beta, vitamin D3 receptor, alpha-tocopherol transfer protein, serum albumin, carbonic anhydrase 2, which relate to visual, nervous, immune, reproductive, skeletal and basal metabolism. Among them, retinoic acid binding protein, retinol-binding protein, transthyretin, vitamin D3 receptor and carbonic anhydrase 2 were closely associated with bone metabolism, which suggest that macamides were anti-osteoporosis active substances.(3) The effect of N-benzyl-palmitamide on promoting bone formation were confirmed on cell model. By MC3T3-E1 Subclone 14 cell, the effects of N-benzyl-palmitamide on osteoblast were researched. The result shown that N-benzyl-palmitamide could promote MC3T3-E1 cell proliferation, differentiation and mineralization, showed promoting bone formation, which might through increasing the expression levels of estrogen receptor and the key factor of osteogenesis, such as BMP-2, Cbfal, COL1 and ALP.(4) The effects of N-benzyl-palmitamide on anti-osteoporosis were further confirmed on animal model. By constructing retinoic acid induced osteoporosis rats model, the effects of N-benzyl-palmitamide on anti-osteoporosis were researched. The results shown that N-benzyl-palmitamide could improve bone mineral density of femur and spine, increasing bone ash and calcium content, improve bone morphological structure, showed anti-osteoporosis effect. N-benzyl-palmitamide might regulate bone metabolism by promoting the ALP activity, resisting decline in estrogen levels and increase in TRAP activity caused by retinoic acid.(5) The effects of maca alcohol extract containing N-benzyl-palmitamide and N-benzyl-palmitamide were compared. The result shown the effects of extract in promoting osteoblast proliferation, improve the density of femur and spine, and improve bone physical parameters and morphological structure were similar to N-benzyl-palmitamide alone, while in promoting ALP activity and improve bone ash and bone calcium content significantly superior to N-benzyl-palmitamide alone. N-benzyl-palmitamide in maca is a important anti-osteoporosis ingredients, but also contain other has certain anti-osteoporosis effect or synergy. In the experiment using doses, maca alcohol extract and N-benzyl-palmitamide were not exhibit obvious adverse reactions, such as uterine weight.In conclusion, N-benzyl-palmitamide were screened out as an active constituent of maca in preventing osteoporosis by using virtual screening against estrogen receptor, the effect of which were verified on osteoblasts and animal. N-benzyl-palmitamide might regulate bone metabolism by estrogen signaling pathways, BMP-2 signaling pathways and retinoic acid signaling pathways.更多还原